Soluble solids modification using sucrose phosphate synthase encoding sequences

ABSTRACT

This invention relates to methods for the utilization of sucrose phosphate synthase encoding sequences to modify the soluble solids in plant sink tissue.

TECHNICAL FIELD

The present invention is directed to compositions and methods related tomodification of the sucrose biosynthesis pathway to alter the solublesolids content in selected plant tissue.

BACKGROUND

With the development of genetic engineering techniques, it is nowpossible to transfer genes from a variety of organism into the genome ofa large number of different plant species. This process has manyadvantages over plant breeding techniques, as genes may now betransferred from one plant species to another plant species, rather thansimply from a plant to the same, or different, but closely related,species.

Sucrose is one of the primary end products of photosynthesis in higherplants. It is also the major carbohydrate transported to sucroseaccumulating, or carbon sink, tissues for plant growth and development(Pate, (1976) In: Transport and Transfer Processes in Plants, pp.253-289, Wardlaw, J. F., Passioura, J. B., eds. Academic Press, NewYork). Plant regions, such as leaf tissue, where sucrose is synthesizedare commonly referred to as sucrose source tissue. Plant storage organs,such as roots or tubers, and fruits are examples of sink tissues.

Sucrose phosphate synthase (SPS) cDNA sequence, SPS constructs andtransgenic SPS tomato lines are described in co-pending application,Ser. No. 08/175,471. This information is also published in Worrell etal. (The Plant Cell (1991) 3:1121-1130), incorporated herein byreference. In particular, the co-pending application describes a maizeSPS under the control of the Rubisco small subunit promoter (SSUpromoter) from tobacco, providing preferential expression in leaftissue. Maximum activity of SPS is shown to be significantly increasedin leaves of tomato plants expressing the maize SPS, and the absolutelevels of starch and sucrose in the leaves are altered in the predictedmanner.

Relevant Literature

SPS encoding sequences and the generation of transgenic tomato linesfrom the pCGN3812 SSU-SPS construct is described by Worrell et al. (ThePlant Cell (1991) 3:1121-1130).

Galtier et al. (Plant Physiol. (1993) 101:535-543) examined thephotosynthetic characteristics of SPS transformants.

In PCT Application WO 94/00563 antisense potato SPS is placed behind atuber promoter and used to alter the sucrose levels in potato.

Acid invertase encoding sequences are described by Klann et. al., (PlantPhys. (1992) 99:351-353.)

SUMMARY OF THE INVENTION

By this invention, a method is disclosed whereby a construct encodingsucrose phosphate synthase (SPS) can be used to modify the solidscontent of plant sink tissue. Soluble solids include simple sugars.Total solids includes more complex carbon compounds, such as starchesand cellulose. In one embodiment of the present invention, methods aredisclosed for increasing the sweetness of fruit.

The method provided for increasing the total solids in a plant sinktissue will modify total solids from a given ratio of total solids perunit weight of sink tissue, as measured in control plant cells, to adifferent ratio of total solids per unit weight of sink tissue. Theamount of sucrose available to growing tissues in the plant isincreased, and the increased sucrose results in increased total solidsper unit weight in the sink tissues of the plant. The method generallycomprises growing a plant having integrated into its genome a constructcomprising as operably linked components in the 5' to 3' direction oftranscription, a transcription initiation region functional in a plantcell and a DNA encoding SPS.

The construct transcription initiation region may be constitutive ortissue specific, i.e., preferentially expressed and functional in cellsof a particular plant tissue, for instance fruit or leaf. Many suchtissue specific promoter regions are known, such as the Rubisco smallsubunit promoter preferentially expressed in leaf tissue, the patatinpromoter expressed in potato tubers and the E8 promoter specific forfruit tissue.

In one embodiment the method produces sink tissue having increasedcarbon as soluble solids, as an increased ratio of soluble solids perunit weight of sink tissue, as compared to that measured in controlplant cells. This results from the increased levels of sucrosegenerating an increased rate of transportation of the available sucroseinto the carbon sink tissue.

In another embodiment a method is provided for modifying the solublesolids ratios in sink tissue, such as the ratio of sucrose to fructose,as compared to that measured in control plant cells or tissue. If theincreased soluble solids in said sink tissue comprises fructose, aphenotype will result having an increased sweetness as compared to thecontrol tissue. Methods are also disclosed, however, whereby a decreasedratio of fructose to sucrose, and whereby a reduced sweetness phenotypemay be produced.

The use of constructs comprising encoding sequences to other sucrosemetabolizing enzymes, such as acid invertase, or the utilization of suchenzymes which are endogenous to the plant sink cells, can be usedadvantageously with this invention. For instance, acid invertase can beexpressed in the cells of sink tissue from an expression construct, or,alternatively, the sink tissue can be prevented from converting sucroseto fructose and glucose by the use of an antisense acid invertaseconstruct, whereby cells of the sink tissue will have a decreased acidinvertase activity, and thereby a decreased ratio of fructose to sucroseas compared to cells in a control sink tissue.

Considered part of this invention are the plants, plant cells and plantsink tissue transformed by nucleic acid sequences to SPS demonstrating amodified carbon solids content. The modification can be an increase inone or more solids components, or a change to the ratios of solidscomponents. In a particular embodiment, the invention provides fruithaving increased total soluble solids and/or modified or increasedfructose levels, as measured per unit weight. A preferred embodimentincludes fruit having a modified sweetness phenotype from an increasedratio of fructose to sucrose in the soluble solids.

Transformed plant cells of this invention are obtainable through planttransformation techniques which utilize Agrobacterium to transfer DNA tothe plant cells or alternatively through direct transfer techniques suchas electroporation, microinjection or DNA bombardment. In either case,plant carbon sink tissue, such as fruit, may be obtained which hasincreased or decreased soluble solids content and/or modified sweetnessproperties.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a method for producing plant cellsdemonstrating modified soluble solids content. The method utilizes a DNAsequence encoding SPS integrated in the cellular genome as the result ofgenetic engineering. Plant fruit which contains increased levels ofsoluble solids and an altered sweetness phenotype is also contemplatedin this invention. The gene encoding SPS is described in copendingapplication Ser. No. 08/175,471, filed on Dec. 27, 1993.

The mechanism whereby the expression of exogenous SPS modifies carbonrelationships derives from source-sink relationships. For instance, inthose situations where SPS is expressed in leaves, plants have highersugar and less starch in the leaves. The leaf tissue is a sucrosesource, and if this higher sucrose is transported to a sink it resultsin increased storage carbon (sugars, starch, etc.) per given weight ofthe sink tissue.

The increased soluble solids in transgenic tomato lines transformed withSPS was unexpectedly shown to be glucose and fructose, not sucrose.Thus, when the increased carbon is in the form of soluble solids, theincrease may be in glucose and fructose levels as an alternative to, oras well as in, changes to levels of sucrose. In the tomato fruit glucoseand fructose are produced from sucrose due to a vacuolar acid invertasethat comes on during ripening. Acid invertase converts sucrose toglucose and fructose, and is known to affect fruit sweetness (Hubbard etal. (1991) Physiol. Plant. 82:191-196). It was not known prior to theinvention whether an increase flow of sucrose into the fruit of tomatowould be converted by exogenous acid invertase levels in this manner.

Fructose is twice as sweet, on a molar basis, as glucose, thus theinvention provides a mechanism by which to selectively increase fructosein fruits for increasing sweetness. Thus, in one embodiment thisinvention produces tomato fruit (a sink tissue) having increasedsweetness.

Thus, the method of altering sink tissue solids with sequences to SPSmay be advantageously used in conjunction with endogenous sucrosemetabolizing enzymes, whereby sequences to SPS are employed as the soletransformed encoding sequence. The method may also be employed inconjunction with other transformed sequences, for instance sequencesencoding other sucrose metabolizing enzymes. Inhibition of certainsucrose metabolizing enzymes could result from the use of antisenseexpression. The inhibition of acid invertase in tomato fruit, forinstance, can lead to fruit having elevated levels of sucrose in thetomato fruit. The sequence to acid invertase is known (Klann et al.,(1992) Plant. Phys. (1992) 99:351-353). Expression of other sucrosemetabolizing enzymes may result in alterations to other carboncomponents, for instance the expression of starch synthesizing enzymesto act in concert with the increase availability of sucrose may resultin increased starch levels in the sink tissue. Such enzymes are known tothe art, with many described in co-pending application having the Ser.No. 08/016,881, the teachings of which are incorporated herein byreference.

A sucrose metabolizing enzyme considered in this invention includes anysequence of amino acids, such as protein, polypeptide, or peptidefragment, which demonstrates the ability to catalyze a reaction involvedin the synthesis or degradation of sucrose or a precursor of sucrose.These can be endogenous plant sequences, by which is meant any sequencewhich can be naturally found in a plant cell, including native(indigenous) plant sequences as well as sequences from plant viruses orplant pathogenic bacteria, such as Agrobacterium or Rhizobium speciesthat are naturally found and functional in plant cells.

It will be recognized by one of ordinary skill in the art that sucrosemetabolizing enzyme sequences may also be modified using standardtechniques of site specific mutation or PCR, or modification of thesequence may be accomplished in producing a synthetic nucleic acidsequence and will still be considered a sucrose biosynthesis enzymenucleic acid sequence of this invention. For example, wobble positionsin codons may be changed such that the nucleic acid sequence encodes thesame amino acid sequence, or alternatively, codons can be altered suchthat conservative amino acid substitutions result. In either case, thepeptide or protein maintains the desired enzymatic activity and is thusconsidered part of the instant invention.

A nucleic acid sequence to a sucrose metabolizing enzyme may be a DNA orRNA sequence, derived from genomic DNA, cDNA, mRNA, or may besynthesized in whole or in part. The structural gene sequences may becloned, for example, by isolating genomic DNA from an appropriatesource, and amplifying and cloning the sequence of interest using apolymerase chain reaction (PCR). Alternatively, the gene sequences maybe synthesized, either completely or in part, especially where it isdesirable to provide plant-preferred sequences. Thus, all or a portionof the desired structural gene may be synthesized using codons preferredby a selected plant host. Plant-preferred codons may be determined, forexample, from the codons used most frequently in the proteins expressedin a particular plant host species. Other modifications of the genesequences may result in mutants having slightly altered activity. Onceobtained, a sucrose metabolizing enzyme may be utilized with the SPSsequence in a variety of ways.

Other endogenous plant sequences may be useful in nucleic acidconstructs of this invention, for example to provide for transcriptionof the sucrose metabolizing enzyme sequences. Transcriptional regulatoryregions are located immediately 5' to the DNA sequences of the gene ofinterest, and may be obtained from sequences available in theliterature, or identified and characterized by isolating genes having adesirable transcription pattern in plants, and studying the 5' nucleicacid sequences. Numerous transcription initiation regions which providefor a variety of constitutive or regulatable, e.g. inducible, expressionin a plant cell are known. Among sequences known to be useful inproviding for constitutive gene expression are regulatory regionsassociated with Agrobacterium genes, such as for nopaline synthase(Nos), mannopine synthase (Mas), or octopine synthase (Ocs), as well asregions coding for expression of viral genes, such as the 35S and 19Sregions of cauliflower mosaic virus (CaMV). The term constitutive asused herein does not necessarily indicate that a gene is expressed atthe same level in all cell types, but that the gene is expressed in awide range of cell types, although some variation in abundance is oftendetectable.

In providing for transcription and/or expression of the sucrosemetabolizing enzyme sequences, for various reasons one may wish to limitthe expression of these enzymes to plant cells which function as carbonsinks. Towards this end, one can identify useful transcriptionalinitiation regions that provide for expression preferentially inspecific tissue types, such as roots, tubers, seeds or fruit. Thesesequences may be identified from cDNA libraries using differentialscreening techniques, for example, or may be derived from sequencesknown in the literature. Useful transcriptional initiation regionspreferentially provide for transcription in certain tissues or undercertain growth conditions, such as those from napin, seed or leaf ACP,the small subunit of RUBISCO, patatin, zein, and the like. Fruitspecific promoters are also known, one such promoter is the E8 promoter,described in Deikman et al. (1988) EMBO J. 2:3315-3320; and DellaPennaet al. (1989) Plant Cell 1:53-63, the teachings of which areincorporated herein by reference. An E8-SPS construct (fruit-specificpromoter) will express SPS in a fruit-specific manner, whereby thelevels of sucrose produced in the fruit may be elevated. If coupled withantisense invertase, the increase in sucrose would be maintained. Thisis a particular issue in tomatoes where acid invertase present in thefruit drives the production of glucose and fructose from sucrose.

Sequences to be transcribed are located 3' to the plant transcriptioninitiation region and may be oriented, in the 5'-3' direction, in thesense orientation or the antisense orientation. In the senseorientation, an mRNA strand is produced which encodes the desiredsucrose metabolizing enzyme, while in antisense constructs, an RNAsequence complementary to an enzyme coding sequence is produced. Thesense orientation is desirable when one wishes to produce the sucrosemetabolizing enzyme in plant cells, whereas the antisense strand may beuseful to inhibit production of a related plant sucrose metabolizingenzymes. The presence of sucrose metabolizing enzyme sequences in thegenome of the plant host cell may be confirmed, for example by aSouthern analysis of DNA or a Northern analysis of RNA sequences or byPCR methods.

In addition to sequences providing for transcriptional initiation in aplant cell, also of interest are sequences which provide fortranscriptional and translational initiation of a desired sequenceencoding a sucrose metabolizing enzyme. Translational initiation regionsmay be provided from the source of the transcriptional initiation regionor from the gene of interest. In this manner, expression of the sucrosemetabolizing enzyme in a plant cell is provided. The presence of thesucrose metabolizing enzyme in the plant host cell may be confirmed by avariety of methods including a immunological analysis of the protein(e.g. Western or ELIZA), as a result of phenotypic changes observed inthe cell, such as altered soluble solids content or by assay forincreased enzyme activity, and the like.

Other sequences may be included in the nucleic acid construct providingfor expression of the sucrose metabolizing enzymes ("expressionconstructs") of this invention, including endogenous plant transcriptiontermination regions which will be located 3' to the desired sucrosemetabolizing enzyme encoding sequence. For instance, transcriptiontermination sequences derived from a patatin gene may be utilized whenthe sink tissue is potato tubers. Transcription termination regions mayalso be derived from genes other than those used to regulate thetranscription in the nucleic acid constructs of this invention.Transcription termination regions may be derived from a variety ofdifferent gene sequences, including the Agrobacterium, viral and plantgenes discussed above for their desirable 5' regulatory sequences.

Further constructs are considered which provide for transcription and/orexpression of more than one sucrose metabolizing enzyme. For example,one may wish to provide enzymes to plant cells of the sink tissue whichprovide for modification of the type of soluble solids to be producedtherein, as well as for enhancing or otherwise modifying the increase ordecrease in overall soluble solids production. An example of enzymeswhich may prove useful in modifying soluble solids ratios is the acidinvertase enzyme.

In developing the nucleic acid constructs of this invention, the variouscomponents of the construct or fragments thereof will normally beinserted into a convenient cloning vector, e.g. a plasmid, which iscapable of replication in a bacterial host, e.g. E. coli. Numerousvectors exist that have been described in the literature, many of whichare commercially available. After each cloning, the cloning vector withthe desired insert may be isolated and subjected to furthermanipulation, such as restriction, insertion of new fragments ornucleotides, ligation, deletion, mutation, resection, etc. so as totailor the components of the desired sequence. Once the construct hasbeen completed, it may then be transferred to an appropriate vector forfurther manipulation in accordance with the manner of transformation ofthe host cell.

The constructs of this invention providing for transcription and/orexpression of sucrose metabolizing enzyme sequences of this inventionmay be utilized as vectors for plant cell transformation. The manner inwhich nucleic acid sequences are introduced into the plant host cell isnot critical to this invention. Direct DNA transfer techniques, such aselectroporation, microinjection or DNA bombardment may be useful. To aidin identification of transformed plant cells, the constructs of thisinvention may be further manipulated to include plant selectablemarkers. The use of plant selectable markers is preferred in thisinvention as the amount of experimentation required to detect plantcells is greatly reduced when a selectable marker is expressed. Usefulselectable markers include enzymes which provide for resistance to anantibiotic such as gentamycin, hygromycin, kanamycin, and the like.Similarly, enzymes providing for production of a compound identifiableby color change, such as GUS, or luminescence, such as luciferase areuseful.

An alternative method of plant cell transformation employs plant vectorswhich contain additional sequences which provide for transfer of thedesired sucrose metabolizing enzyme sequences to a plant host cell andstable integration of these sequences into the genome of the desiredplant host. Selectable markers may also be useful in these nucleic acidconstructs to provide for differentiation of plant cells containing thedesired sequences from those which have only the native geneticmaterial. Sequences useful in providing for transfer of nucleic acidsequences to host plant cells may be derived from plant pathogenicbacteria, such as Agrobacterium or Rhizogenes, plant pathogenic viruses,or plant transposable elements.

When Agrobacterium is utilized for plant transformation, it may bedesirable to have the desired nucleic acid sequences bordered on one orboth ends by T-DNA, in particular the left and right border regions, andmore particularly, at least the right border region. These borderregions may also be useful when other methods of transformation areemployed.

Where Agrobacterium or Rhizogenes sequences are utilized for planttransformation, a vector may be used which may be introduced into anAgrobacterium host for homologous recombination with the T-DNA on theTi- or Ri-plasmid present in the host. The Ti- or Ri- containing theT-DNA for recombination may be armed (capable of causing gallformation), or disarmed (incapable of causing gall formation), thelatter being permissible so long as a functional complement of the virgenes, which encode transacting factors necessary for transfer of DNA toplant host cells, is present in the transformed Agrobacterium host.Using an armed Agrobacterium strain can result in a mixture of normalplant cells, some of which contain the desired nucleic acid sequences,and plant cells capable of gall formation due to the presence of tumorformation genes. Cells containing the desired nucleic acid sequences,but lacking tumor genes can be selected from the mixture such thatnormal transgenic plants may be obtained.

In a preferred method where Agrobacterium is used as the vehicle fortransforming host plant cells, the expression or transcription constructbordered by the T-DNA border region(s) will be inserted into a broadhost range vector capable of replication in E. coliand Agrobacterium,there being broad host range vectors described in the literature.Commonly used is pRK2 or derivatives thereof. See, for example, Ditta,et al., (Proc. Nat. Acad. Sci., U.S.A.(1980) 77:7347-7351) and EPA 0 120515, which are incorporated herein by reference. Alternatively, one mayinsert the sequences to be expressed in plant cells into a vectorcontaining separate replication sequences, one of which stabilizes thevector in E. coli, and the other in Agrobacterium. See, for example,McBride and Summerfelt (Plant Mol. Biol. (1990) 14:269-276), wherein thepRiHRI (Jouanin, et al., Mol. Gen. Genet. (1985) 201:370-374) origin ofreplication is utilized and provides for added stability of the plantexpression vectors in host Agrobacterium cells.

Utilizing vectors such as those described above, which can replicate inAgrobacterium is preferred. In this manner, recombination of plasmids isnot required and the host Agrobacterium vir regions can supplytrans-acting factors required for transfer of the T-DNA borderedsequences to plant host cells.

In general, the plant vectors of this invention will contain sucrosemetabolizing enzyme sequence(s) and sequences providing fortranscription or expression of these sequences in a plant host cell. Theplant vectors containing the desired sequences may be employed with awide variety of plant cells and plants. Fruit producing plants andplants which produce and store reserve starch are of particularinterest, the latter including, but in no way limited to, corn, cerealgrains, sorghum, rice, potato, tapioca, cassava, arrowroot and sago. Themethod may also be useful in increasing the sweetness in fruits,including, but in no way limited to, tomato, strawberry and melon.

Also considered part of this invention are plants containing the nucleicacid sequences of this invention, and following from that, plantsmodified by the expression of SPS and possibly additional sucrosemetabolizing enzymes as the result of expression of the sequences inplant cells, or having a decreased expression of a such by theinhibition of such additional sucrose metabolizing enzymes fromantisense expression of the encoding sequences Methods of regeneratingwhole plants from plant cells are known in the art, and the method ofobtaining transformed and regenerated plants is not critical to thisinvention. In general, transformed plant cells are cultured in anappropriate medium, which may contain selective agents such asantibiotics, where selectable markers are used to facilitateidentification of transformed plant cells. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be used toestablish repetitive generations either from seed or using vegetativepropagation techniques.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

Measurement of specific gravity or free sugar content may be useful todetect modified sweetness or total soluble solids, with other methods,such as HPLC and gel filtration, also being useful.

EXAMPLES

Constructs and plants with the designation 3812 are as described inco-pending application Ser. No. 08/175,471, which in turn is acontinuation of U.S. patent application Ser. No. 07/672,646, both ofwhich are incorporated by reference. Briefly, the construct pCGN3812contains the construct 5'-35S-nptII-tml-3'/5'=tobacco Rubisco SSU-maizeSPS cDNA-3'. Tomato lines arising from separate transformation eventsare signified by a hyphen and a number following the constructdesignation.

Example 1

Soluble Solids in Fruit of T2 Plants

Investigation of the soluble solids in the fruits of the SSU-SPS lineswas initially done on extracts from fruit of 3812-9 and 3812-11 linesgrown in a Biotron incubator. T2 plants were illuminated by metal halidelamps at a peak level of 500 umol photons/m/s (pot level), 26 C. for the16 h day and 18 C. at night, and a relative humidity of 60%. Plants werewatered daily with half-strength Hoagland's solution (Hoagland andArnon, Calif. Agricult. Exp. Sta. Cir. (1938) 357:1-39). These lineswere segregating as the original lines contained at least 2 insertions.

Brix analysis (soluble solids) on extracts from these plants revealedlines with Brix readings as much as 40% higher than the controls. Theextracts measured were the average of 3 fruit from one plant.

Measurements were also taken for fruit from a segregating T2 populationof 3812-11 plants in the greenhouse. The controls averaged a Brixreading of 3.5 while the transgenics averaged 4.0, an increase of 14%.

Example 2

Measurements on Homozygous Fruit

T4 homozygous lines were generated from original 3812-9 transformants inUC82-B tomatoes. The original line segregated 15:1 for Kan resistance,indicating that is it had two insertion sites. Two homozygous lines weregenerated and verified to be different by Southern border analysis.These lines were designated A and B.

Individual homozygote (T4) lines were grown in the greenhouse, withthree fruit taken from each plant and 3 plants analyzed from each line.The Brix of the UC82B controls was 3.35 while the Brix on the 3812-9lines ranged from 3.7 to 4.1. This is an increase from 12% to 24%.Statistics (LSD) on all the lines in which fruit from 3 plants wereanalyzed showed these results to be significant at a 0.01% level (99%).

Example 3

Brix Analysis of Field Trial Fruit

Field trial results of RI measurements are provided in Table 1. The R/I(refractive index) was measured several times on the fruit of theseplants (Table 1). R/I is a measure of soluble solids and is indicativeof sugars and acids. The transgenic A lines consistently had a higherR/I (3.9 to 4.1) than the control. (3.5).

                  TABLE 1                                                         ______________________________________                                        Summary of Refractive Index Measurements                                                                              Overall                               Line    Reading I                                                                              Reading 2                                                                              Reading 3                                                                            Reading 4                                                                            Aug                                   ______________________________________                                        Trial 1                                                                       UC82-B  3.3      3.9      3.6    3.0    3.5                                   A Lines (X2)                                                                          4.0      4.4      4.6    3.8    4.2                                   Trial 2                                                                       UC82-B  4.1      4.1      3.9           4.0                                   A Lines (X3)                                                                          4.8      4.3      4.2           4.4                                   B Lines (X2)                                                                          4.4      4.4      4.2           4.3                                   Trial 3                                                                       UC82B   3.2                                                                   A Lines (X1)                                                                          4.2                                                                   B Lines (X1)                                                                          4.2                                                                   ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    Sugars of Tomatoes of plants transformed with SPS Gene                                        Sugar concentration (%)                                                                       Relative increase                             ID # Tomato line                                                                           RI Sucrose                                                                           Glucose                                                                           Fructose                                                                           Total                                                                            over control                                  __________________________________________________________________________    41000 #1                                                                           Control 3.9                                                                              0.08                                                                              1.33                                                                              1.62 3.03                                             41000 #2                                                                           Control 4.2                                                                              0.11                                                                              1.51                                                                              1.75 3.37                                             41003                                                                              SSU-SPS-A-75-5                                                                        4.9                                                                              0.19                                                                              1.58                                                                              2.58 4.35                                                                             36%                                           41004                                                                              SSU-SPS-A-91-4                                                                        4.9                                                                              0.19                                                                              1.61                                                                              2.55 4.35                                                                             36%                                           41008                                                                              SSU-SPS-B-87-2                                                                        4.6                                                                              0.22                                                                              1.59                                                                              2.37 4.18                                                                             31%                                           Average increase                                                                           0.18                                                                             0.10                                                                              0.17                                                                              0.81 1.09                                                                             34%                                           due to SPS                                                                    __________________________________________________________________________

Example 4

HPLC Analysis on SSU-SPS Fruit Sugars

Fruit from the SPS plants described in Example 3 were further analyzedby HPLC to determine contributions of individual sugars to the increasedsoluble solids content. As seen in Table 2, sucrose did not increase asmuch as might be expected based on the fact that sucrose is the sugartransported by the plant into the fruit. Glucose was not increased asmuch as fructose, which increased nearly 50%.

It is evident from the above results, that plant cells and plants can beproduced which have improved properties or may produce a desiredphenotype. In accordance with the subject invention, it is now seen thatSPS sequences may be introduced into a plant host cell and be used toexpress the enzyme to increase soluble solids content in fruit.Moreover, it is seen that the SPS may be used to alter the overallcontent and ratio of soluble solids in plant sink tissue, resulting in ademonstrable phenotype in planta, such as altered fruit sweetness. Inthis manner, fruits, such as tomato fruit, having modified sweetness maybe obtained.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teaching of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method of modifying the soluble solids ratiosin a cell of plant sink tissue, as compared to that measured in controlplant cells, to a different ratio of soluble solids, wherein said methodcomprises:growing a plant having acid invertase in cells of said sinktissue, said plant having integrated into its genome an expressionconstruct comprising, as operably linked components a transcriptioninitiation region which is functional in a plant cell and a DNA encodinga sucrose phosphate synthase derived from corn which is functional insaid plant cell, wherein said DNA encoding a sucrose phosphate synthaseis not naturally linked to said transcription initiation region, andwherein said cell is grown under conditions which permit saidtranscription initiation region to function, whereby an increased levelof sucrose is made available to said sink tissue where it is acted on bysaid acid invertase to modify the soluble solids in said sink tissue ascompared to a sink tissue of said control plant cells.
 2. The method ofclaim 1 wherein cells of said sink tissue have an increased ratio offructose to sucrose as compared to that of a cell in a control sinktissue.
 3. The method of claim 1 wherein said transcription initiationregion is tissue specific.
 4. The method of claim 1 wherein saidtranscription initiation region is functional in a fruit cell.
 5. Themethod of claim 1 wherein said transcription initiation region isfunctional in a leaf cell and wherein said sucrose is transported intosaid sink tissue.
 6. The method of claim 5 wherein said transcriptioninitiation region is a Rubisco small subunit promoter.
 7. A sink tissueplant cell having modified levels of fructose and sucrose, wherein saidmodified levels of fructose and sucrose are produced according to themethod of claim
 1. 8. The method of claim 1 wherein said acid invertaseis endogenous to said cells of said sink tissue.
 9. A method ofmodifying the ratio of soluble solids in a plant sink tissue, saidmethod comprising:growing a transgenic plant having integrated into itsgenome (1) a first expression construct comprising as operably linkedcomponents, a first transcription initiation region functional in aplant cell and DNA encoding a sucrose phosphate synthase derived fromcorn which is functional in said cell, wherein said DNA encoding saidsucrose phosphate synthase is not naturally linked to said firsttranscription initiation region, and either (2) a second expressionconstruct comprising as operably linked components, a sinktissue-specific second transcription initiation region functional in acell of said sink tissue and DNA encoding an acid invertase which isfunctional in said cell of said sink tissue, wherein said DNA encodingsaid acid invertase is not naturally linked to said second transcriptioninitiation region, or (3) a third expression construct comprising athird transcription initiation region functional in a plant cell and DNAencoding an antisense acid invertase sequence, wherein said growing isunder conditions which permit expression of said DNA, and wherein saidfirst, second and third transcription initiation regions may be the sameor different, whereby the ratio of soluble solids in said plant sinktissue is modified as compared to a sink tissue of a control plant. 10.The method of claim 9 wherein said transgenic plant comprises said firstand second expression constructs and said sink tissue comprises anincreased ratio of fructose to sucrose as compared to a sink tissue ofsaid control plant.
 11. The method of claim 9 wherein said transgenicplant comprises said first and third expression constructs and said sinktissue contains a decreased ratio of fructose to sucrose as compared toa sink tissue of said control plant.
 12. The method of claim 11 whereinsaid third expression construct comprises a double 35S promotertranscription initiation region.
 13. A method of modifying the ratio ofsoluble solids in a tomato plant sink tissue, said methodcomprising:growing a tomato plant having acid invertase in cells of saidsink tissue, said tomato plant having integrated into its genome anexpression construct comprising as operably linked components, atranscription initiation region which is functional in a plant cell anda DNA encoding a sucrose phosphate synthase derived from corn which isfunctional in said cell, wherein said DNA encoding a sucrose phosphatesynthase is not naturally linked to said transcription initiationregion, and wherein said cell is grown under conditions which permitsaid transcription initiation region to function, whereby an increasedlevel of sucrose is made available to said sink tissue where it is actedon by said acid invertase to modify the soluble solids in said sinktissue as compared to a sink tissue of a control tomato plant.
 14. Amethod of modifying the ratio of soluble solids in a tomato plant sinktissue, said method comprising:growing a tomato plant having integratedinto its genome a first expression construct comprising as operablylinked components, a first transcription initiation region functional ina plant cell and DNA encoding a sucrose phosphate synthase derived fromcorn, wherein said DNA encoding said sucrose phosphate synthase is notnaturally linked to said first transcription initiation region, and asecond expression construct comprising as operably linked components, asink-tissue specific second transcription initiation region which isfunctional in a cell of said sink tissue and DNA encoding an acidinvertase, wherein said DNA encoding said acid invertase is notnaturally linked to said second transcription initiation region, andwherein said growing is under conditions which permit expression of saidDNA so that the ratio of soluble solids in said sink tissue is modifiedas compared to a sink tissue of a control tomato plant.
 15. A method ofmodifying the ratio of soluble solids in a tomato plant sink tissue,said method comprising:growing a tomato plant having integrated into itsgenome a first expression construct comprising as operably linkedcomponents, a first transcription initiation region functional in aplant cell and DNA encoding a sucrose phosphate synthase derived fromcorn, wherein said DNA encoding said sucrose phosphate synthase is notnaturally linked to said first transcription initiation region, and asecond expression construct comprising as operably linked components asecond transcription initiation region functional in a cell of said sinktissue and DNA encoding an antisense acid invertase sequence, whereinsaid growing is under conditions which permit expression of said DNA sothat the ratio of soluble solids in said sink tissue is modified ascompared to a sink tissue of a control tomato plant.